Use of anti-ferritin monoclonal antibodies in the treatment of some cancers (divisional)

ABSTRACT

The invention concerns the use, in the preparation of a medicine for treating cancer whereof the cells exhibit overexpression of a product of a gene of the myc-related family, of an anti-ferritin monoclonal antibody or a fragment thereof, said antibody or said fragment identifying an epitope common to acidic and basic human ferritins.

This application is a division of U.S. application Ser. No. 09/952,307filed on Sep. 13, 2001, which is a Continuation of InternationalApplication PCT/FR01/00192 filed on Jan. 19, 2001, which claims priorityto French application 00/00718 filed on Jan. 20, 2000 which applicationsare incorporated herein by reference.

The present invention relates to the provision of novel means for thediagnosis and therapy of cancers characterized by overexpression of geneproducts from the myc family. These means essentially include the use ofmonoclonal antibodies recognising an epitope common to acidic and basicisoferritins.

Ferritin is a protein with a molecular weight of 440000 kDa used tostore iron in cells. Since 1942, the date at which GRANICK isolatedhepatic ferritin and until recently, this molecule was considered to bea uniquely cellular iron storage protein mainly located in the liver,the spleen and the bone marrow. It only appears in the serum in theevent of a substantial overload of iron or during hepatic necrosis withliberation of this tissue protein into the vascular space. Moresensitive detection methods using radioactive, enzymatic or fluorescenttracers have demonstrated the existence of a base concentration of serumferritin and have allowed the study of variations thereof during thecourse of various diseases. At the same time, a number of research teamshave endeavoured:

-   -   to refine knowledge regarding the biochemical structure of        tissue ferritin;    -   to discover the reasons for the heterogeneity of the molecular        forms grouped under the term “isoferritins”;    -   to compare the different characteristics of ferritin extracted        from plasma or from different healthy or diseased organs.

Current data show that ferritin is a macromolecular structureconstituted by a glycoprotein structure in the form of a shell,Apoferritin, which is the protein component and which encloses ametallic core of iron atoms distributed in iron crystals inside itscavity.

X ray crystallographic analysis of ferritin has shown the presence of 24sub-units, distributed in a rigorous symmetry. The sub-units are of twotypes, H and L.

The genes coding for each of the sub-units have been cloned and theirchromosomal location is known (Murow H. N., Aziz N., Leibold E. A. etal., The ferritin gene's expression and regulation, Ann. NY Acad. Sci.1988, 528 113). The H type sub unit, with a molecular weight of 21000,is in the majority among the most acidic isoferritins such as ferritinextracted from HeLa cells or ferritin of cardiac origin (H=heart), butit is also present to a lesser extent in the liver and spleen. Thesecond type of sub-unit (L), with a molecular weight of 16000, is morespecifically in the majority in the most basic molecules of hepaticorigin (L=liver) or originating in the spleen.

The iron stored in ferritin is mobilised for the synthesis of proteinsthat incorporate ferrous or ferric ions as functional components(haemoglobins and enzymes such as cytochromes and catalases). Theprincipal functions of ferritin in the liver, spleen and marrow arefirstly to protect tissues from oxidation and damage caused by freeradicals produced by interactions with iron, water and oxygen, andsecondly to re-use iron for the synthesis of haemoglobins. In otherorgans or in plasma, ferritin contains little or no iron and itsfunction is poorly understood.

It has long been known that different ferritin phenotypes exist(isoferritins) (Drysdale J. W. (1977), Structure and metabolism, (CibaFoundation Symposium 51, New Series), pages 41-57. In 1965, Richterdemonstrated that ferritin prepared from human liver and carcinomatousepidermoid HeLa cells had different electrophoretic migration rates,that could not be explained by differences in the iron content. Intotal, about twenty isoferritins were characterized.

Techniques such as electrofocussing, ion exchange chromatography andtwo-dimensional electrophoresis under denaturing conditions showed thatisoferritins are formed by variable combinations of the two sub-units Hand L (Murow et al., supra).

The role of basic isoferritins is well known, particularly that of serumferritin in phenomena directly linked to iron metabolism. That of acidicferritins is controversial: many authors have used it as a tumoralmarker in man. In 1968, a α2H globulin, later termed α2H isoferritinswas discovered in serum from patients with different neoplasias (BuffeD. et al., Presence d'une protéine d'origine tissulaire α2-Hglobulinedans le serum de sujets atteints d'affections malignes [Presence of aprotein originating from tissue α2H globulin in the serum of subjectswith malignant disorders], Int. J. Cancer (1968) 3: 850-856). Marcus Det al., (J. Natl. Cancer Disti. (1975) 55: 791-795) described anabnormal ferritin in the serum from patients with breast cancer.Drysdale J. et al., (Cancer Res. (1974) 34: 3352-3361) described an acidisoferritin identical to the ferritin contained in HeLa cells with aparticularly high concentration in different types of neoplasia. MorozC. et al., (Clinical Exp. Immunol. (1977) 29: 30-76) describe a factor,which is an isoferritin, secreted by a sub-population of T lymphocytespresent in particular in breast cancer and Hodgkin's disease. Finally,it should be noted that the majority of human neoplastic processes(Hodgkin's disease, breast, ovarian, pancreas, lung cancers,epidermoidal cancers of the head and neck, neuroblastoma, acutelymphoblastic leukaemia (ALL), Kaposi sarcomas, etc. . . . ) areaccompanied by an elevation in serum ferritin. In all cases, a highlevel of serum ferritin is a negative prognostic factor (Hann H. W.,Evans A. E., Siegel S. E. et al., Cancer Res., (1985) 45; 2843-2848;Jacobs A., Slater A., Wittaker J. A. et al., J. Cancer, (1976) 34: 533).

It has also been shown that in several types of cancer, there is a localtumoral increase in tissue ferritin, although the stromal or purelytumoral reactional origin of this excess ferritin has not been formallydefined. Nevertheless, all authors are in agreement that it concernsacidic ferritin.

The development of antibodies and in particular monoclonal antibodies inimaging or for the treatment of certain diseases has been the aim ofmany developments.

A review of these developments was published in 1998 (P. S. Multani etal., (1998), Journal of Clinical Oncology, vol. 11. 16: 3691-3710). Thediagnostic or therapeutic use of antibodies in a cell screening systemwith substances with a selective toxicity to specifically eliminatepathological cells is known as the magic bullet.

Briefly, the different possibilities for using monoclonal antibodies inthis context are as follows:

-   -   a) the use of native antibodies as an immune effector, used in        anti-tumoral therapies with a CDCC (complement-dependent cell        cytotoxicity) activity or an ADCC (antibody-dependent cell        cytotoxicity) activity. The antibody with a certain specificity        as regards this target cell via its Fab fragments can then cause        a cellular immune reaction due to Fc fragments of the same        antibody. It can also involve blocking a receptor of the target        cell or an anti-idiotypic vaccination specific for the tumoral        antigen;    -   b) the antibody can be coupled to a toxin or a drug; the        antibody is then a vector that transports said toxin or drug to        the target;    -   c) antibodies can also act as a galenical vector by coupling        with liposomes or other analogous systems into which cytoxic        substances or drugs or anti-sense oligonucleotides, etc. . . .        can be introduced.    -   d) the antibodies can be directly or indirectly radiolabelled        (chelate or bispecific antibody with an “antichelate, etc” site)        and their radiation can be used for therapeutic ends or for        imaging. A number of possible isotopes exist, in particular for        labelling the antibodies. The most frequently used isotope is        iodine 131. This method has a number of disadvantages (iodine        fixing in the thyroid, existence of endogenous dehalogenases).        Further, iodine 131 does not emit pure β radiation. Finally, its        range length is short (1.1 mm).

Coupling to indium 111 for imaging or yttrium 90 for therapy has alsobeen developed. In that case, coupling is generally carried out bygrafting a chelate to an antibody, the chelate fixing the radioactiveemitter using techniques described and developed by the team headed byS. M. Quadri and H. M Vriesendorp (Vriesendorp H. M. et al., (1991), J.Clin. Oncol. 9: 918-928; P. E. Borchardt et al., (1998), The Journal ofNuclear Medicine 39: 476-484). The choice of yttrium 90 as a therapeuticradio-isotope is pertinent to cancerology. That isotope emits a pure δradiation; its half-life is 67 hours and its range length is 6.6 mm. Itappears to be well adapted to treating solid tumoral masses, inparticular Hodgkin's disease and malign non Hodgkin's lymphomas.Encouraging results have been obtained by the same team (H. M.Vriesendorp et al., (1995), Cancer Research 55, 58-92). These authorsdescribe trials with polyclonal antiferritin antibodies labelled with Y90 in patients with Hodgkin's disease resistant to conventionaltreatment methods combining chemotherapy and external radiotherapy andbone marrow grafts.

One of the biological characteristics of this disease is hyperexpressionof ferritins by tumoral cells and the reactional cells surrounding them.Imaging tumour sites (indium 111) and the treatment of resistant formsof this disease by the system described in Vriesendorp et al., (1995),Cancer Research 55: 58-92 has produced good results.

Further, it is known that the myc oncogene family (the most importantare c, N. L) codes for nuclear proteins that bind to DNA. In a normalcell, transcription of myc genes increases in the hours following amitogenic signal. Proteins coded by the genes from the myc family have a“leucine zipper” moiety. This structure enables the formation ofheterodimers (with c-fos or c-jun) or homodimers. Currently, thefunction of the c-myc product is viewed as being that of regulation ofthe transcription of cellular genes involved in mitosis initiation. Insummary, myc genes acquire oncogenic properties by mechanisms thatresult in an overexpression of their products. This overexpression canbe a result of:

-   -   gene amplification; or    -   activation of gene transcription; or    -   post-transcriptional modifications involving increased stability        of mRNA. In vivo studies on the overexpression of the products        of genes from the myc family in different tissues by the        creation of transgenic animals has confirmed these facts.        Finally, any increase in proliferation is physiologically        accompanied by an increased expression of genes from the myc        family.

The three principal members of the myc family (c, N, L) are among themost frequently found oncogenes in human cancers that are activated bythese three different mechanisms. In particular, c-myc is found inmalignant lymphomas, L-myc and N-myc in small cell lung cancers andneuroblastomas from which the latter has been characterized. In themajority of malign human tumours, the degree of expression of myc genesis a negative prognostic factor.

Known target genes for which transcription is regulated by the productsof genes from the myc family are few. The gene coding for the H chain offerritin is the latest gene for which it has just been shown that it isnegatively regulated by the product of the c-myc gene (Wu et al.,Coordinated regulation of iron controlling genes, H ferritin and IRP2c-myc, Science (1999) 283: 676-679). In other words, expression of thec-myc gene and/or other members of this family in cancer cells isinversely proportional to ferritin expression.

The present invention results from the unexpected and unenvisagedobservation from the available information discussed above whereby afterimmunising mice with a ferritin extracted and purified from a humanspleen, certain monoclonal antibodies had a very high specificity intargeting cancer cells or cancers in which the myc gene isoverexpressed.

The ferritin extraction-purification technique used can obtain two,acidic and basic, forms of ferritin with a high degree of purity. Theextraction-purification technique used in the present inventionparticularly consists of extracting and purifying human ferritinsextracted and purified from patients with microcytic anaemia.Unexpectedly, certain monoclonal antibodies, obtained using theconventional Köhler and Milstein technique, recognise an epitope commonto all acidic and basic, uniquely human isoferritins (J. Kadouche etal., C. R. Acad. Sc. Paris (1982) t. 295 series III, page 443).Monoclonal antibodies are screened in two steps: i) selection ofantiferritin antibodies by capture on a solid base coated with the sameferritin as that used to immunise animals; ii) revealing and fixingspecific antiferritin antibodies fixed to this solid phase by adding thesame ferritin, radiolabelled with iodine 125, to the wells. Selection ofantibodies on a solid phase using purified ferritin and using iodine-125labelled ferritin can select antibodies directed against a common andrepetitive epitope in all human acidic and basic isoferritins. The term“repetitive epitope” means that the same epitope is present a number oftimes in the molecule.

The invention also results from a demonstration whereby, in contrast tothat which could be predicted from the article by Wu et al., (supra),specific monoclonal antibodies for the two forms of ferritin recognisetumour cells or cancerous cells in which the C-myc gene isoverexpressed.

The present invention concerns the use, in the production of a drug forcancer therapy in which cells overexpress the product of a gene from themyc family, of an antiferritin monoclonal antibody or a fragment thereofor a construction including such a fragment, said antibody or saidfragment recognising an epitope that is common to acidic and basic humanferritins.

The term “construction including a fragment of antibody” means a productof a polynucleotide sequence coding said fragment and a polypeptide witha stabilising or transport function, such as albumin, ovalbumin or afragment thereof. It can also mean a conjugate combining the antibody orantibody fragment with an adjuvant and/or a molecule or structureensuring transport and/or stability of said antibody, such as liposomes,cationic vesicles or nanoparticles. Finally, it can mean a combinationof the product of a sequence as described above conjugated with anadjuvant and/or a molecule or structure ensuring transport and/orstabilisation of the molecule.

Therapeutic or diagnostic use in vivo of monoclonal antibodies requiresthat they combine particular qualities of specificity, affinity andnon-toxicity. The term “non-toxicity” means the fact that they do notcause immune reactions or rejections in relation to a substance that canbe administered long-term and in any event repeatedly. The term“specificity” means the fact that the monoclonal antibodies in questiondo not cross-react with antigens other than ferritins.

In terms of specificity, it is important that the antibody recognisesboth acidic isoferritins (specific for neoplastic processes, H form) andbasic isoferritins (martial metabolism, L form).

The antiferritin monoclonal antibody of the invention must also have anaffinity for the corresponding antigen that is sufficient to eliminatethe diffusion of this antibody as much as possible, if necessarycarrying a toxic or therapeutic substance into healthy tissue or cells.In the present invention, the affinity for ferritins must be more than10⁻⁹ mole/litre.

Further, the epitope that is common to acidic and basic isoferritins ispreferably a repetitive epitope. Repetition of the same epitope on theisoferritins allows a number of antibodies to fix to the same moleculeof ferritin. The diagnostic and therapeutic properties of the resultingcoupling products from these antibodies are advantageously improved inthis manner.

The invention also concerns antiferritin monoclonal antibodies fordiagnosis or therapy of cancers or tumour cells in which overexpressionof a gene from the myc family is observed and which has the followingcharacteristics:

-   -   it recognises an epitope that is common to acidic and basic        ferritins and, preferably, it does not recognise non human        ferritins; more preferably, said epitope that is common to        acidic and basic ferritins is a repetitive epitope;    -   its affinity for the antigen is more than 10⁻⁹ mole/litre.

The antibodies of the invention are preferably kappa type IgGimmunoglobulins. However, they can also be IgM immunoglobulins which,because they have a plurality of epitopic recognition sites, can improvetargeting. When an injection is made directly into the lesion in vivo,IgM is preferred in that it has ten antigen binding sites: the yield isthus higher.

The term “diagnosis” means both in vitro immunoscintigraphic diagnosisand any in vivo diagnostic method using monoclonal antibodies as avector for a substance allowing its localisation.

The quality of the monoclonal antibodies used in the invention resultsfirstly from the quality of the antigen used for immunisation andsecondly, from its degree of purity.

The ferritin used to immunise mice in the process for obtainingmonoclonal antibodies originates from a pathological spleen from apatient with Minkowski-Chauffard's syndrome extracted using the modifiedGranick method using ammonium sulphate and cadmium sulphate. Thisextracted and purified ferritin was used in the protocol described inExample 1 below to immunise mice to obtain hybridomas producingmonoclonal antibodies.

Table I below summarises some of the characteristics of the antibodiesobtained after cloning the hybridomas obtained using the purification,extraction and immunisation technique of the invention.

This table indicates the denomination of the monoclonal and polyclonalantibodies tested, their isotypic nature, their affinity for ferritinsand the reactivity to a whole series of ferritins measured byprecipitation using Ouchterlony's immuno-diffusion technique. Theantibodies used in the invention must have a negative reactivity for allnon human cells or tissues, namely horse spleen and LIA (lemia inhibitoractivities), which is not the case for any of the polyclonal antibodies,nor for M211 monoclonal antibody.

A further characteristic of the antibodies used in the invention istheir affinity constant; it appears that among the antibodies in thistable, only B8 and M29 can be used in the invention.

Finally, in a particular implementation, the antibodies of the inventionare directed against a repetitive epitope common to acidic and basicisoferritins. Such antibodies are demonstrated by a sandwich ELISA testas described in Example 2 below and illustrated in FIG. 3. Being able tocarry out such a test confirms that a plurality of antibodies can fix tothe same ferritin molecule. This property endows it with a definiteadvantage as regards their diagnostic or therapeutic use. Monoclonal M29 M 211 M 386 B 8 — — — antibody Polyclonal — — — — F1 F2 F3 antibodyIsotypes Ig G1K Ig G1K Ig G2b K Ig G1 K — — — Affinity 2.3 10⁻¹⁰ 1.310⁻⁸ 2.2 10⁻⁸ 5.1 10⁻⁹ 1.3 10⁻¹¹ 5.9 10⁻¹¹ 4.0 10⁻¹¹ constant (I/mol)Reactivity: human + + + + + + + liver human + + + + + + + spleenhuman + + + + + + + heart human + + + + + + + placentaHeLa + + + + + + + cells Horse − + − − − + + spleen LIA − − − − + + +

In vivo use of murine or animal monoclonal antibodies is, however,limited because of the production by the organism of anti-speciesantibodies rapidly rendering iterative injections ineffective or evendangerous (anaphylaxis, immuno-complex disease, production ofanti-idiotypic antibodies). It is thus preferable to eliminate the Fcportions from these murine antibodies either by humanisation or byreconstitution. The solution consisting of developing chimeralantibodies has often been used (Nature 321 (1986) 326: 522-525). It isnow possible to clone the genes coding for the two chains of themonoclonal antibodies produced by hybridomas, and these can bemanipulated in vitro and then re-introduced into the secreting cells,such as lymphoid lines or the like, after their re-insertion into theselected cells in appropriate expression vectors. New monoclonalantibodies are then constructed with variable regions from mice or ratsand human constant regions. Different types of immunoglobulins can beobtained. Their specificity in all cases will be the same as that fromthe variable regions used, but they will have the effective propertiesof human immunoglobulins that depend on the isotype selected forconstruction.

A second approach consists in reshaping the antibodies (Riechmann etal., (1988), Nature 332: 323-327). Briefly, the starting point for thisapproach is the observation that the contact sites for antibodies withthe antigen epitopes they recognise are located on certain sequences ofthe variable regions termed hypervariable regions. The techniqueconsists in grafting the hypervariable regions of a murineimmunoglobulin termed the complementarity determining region (CDR) ontovariable regions of a human class G immunoglobulin.

Finally, Clackson et al., (1991), Nature 352: 624-628 and the teamheaded by Lemer (Huse et al., (1989), Science, 246: 1275-1281) used PCRto amplify V genes of expressed heavy chains of murine G immunoglobulin.Different libraries were then created from the genomic DNA extractedfrom immunised mouse spleens. A library of vectors carrying variablegene sequences from all of the antibodies was produced. A λbacteriophage was used as an expression vector in E. coli. Thistechnique produced single chain VH domains of the antibodies. Then,single chain Fv fragments were produced, obtained from a VH region,linked to a VK domain via a linker, using PCR. This method could simplyand cheaply produce specific fragments in a non-immunogenic environment.

Finally, the monoclonal antibodies of the invention, whether IgG of IgM,can be monospecific, bi-specific or polyspecific. The geneticmanipulation techniques described above can combine specificitiesfirstly for ferritin and secondly, for a receptor or surface antigenspecific surface for a cell to be targeted.

A variety of possibilities exist for using monoclonal antibodies in themagic bullet concept. The first is to use antibodies as an immuneeffector, employed in anti-tumoral therapies with a CDCC(complement-dependent cell cytotoxicity) activity or an ADCC(antibody-dependent cell cytotoxicity) activity. It may also concernblocking a receptor of the tumour cell or an anti-idiotypic vaccinationspecific for the tumour antigen.

In the use of the invention, the antibody or fragment thereof can becoupled to a molecule X, where X is a toxic molecule, a drug or aprodrug or a nucleotide sequence or a second antibody regardless of itsrecognition specificity.

a) X is a Toxic Molecule:

A first example is the case where X is a radioactive isotope, preferablya β emitter. A number of possibilities exist for coupling radioactiveisotopes to proteins, in particular monoclonal antibodies. A number ofstudies using antiferritin polyclonal antibodies labelled with indium111 (In 111) and yttrium (Y 90) have produced interesting resultsconcerning imaging tumour sites, preferably with In 111, and concerningthe treatment of refractory forms of Hodgkin's disease (Y 90) (P. E.Borchardt et al., (1998), The Journal of Nuclear Medicine, 39, n^(o) 3,page 476).

Whether IgM or IgG, the radioactive isotope is coupled to the antibodyby the chelation techniques described, namely Borchardt et al., (supra).

The choice of yttrium 90 as therapeutic radioelement is particularlypertinent: it is a pure β emitter with a half-life of 67 hours and arange length of 6.6 mm. It appears to be perfectly suited to treatingtumour masses, in particular Hodgkin's disease, lymphomas or cancers ofthe pancreas.

X can also be a toxic A chain ricin or abrin molecule, or A chaindiphtheria toxin. The toxicity of these polypeptides is well known. Theycan be coupled to immunoglobulin by carboxyl type bonds or by any bondtype that can couple proteins together, in particular via their —COOH,—NH₂, —SH residues, etc., which are well known to the skilled person.

b) X is a Cytotoxic Molecule:

Cytotoxic drugs are known, others are being developed. They can blockcell division, either by blocking transcription or by blockingtranslation or by inducing apoptosis. Examples that can be cited aremitomycin, adriamycin, taxol, etc. The problem with using such drugs istheir toxicity towards healthy cells or tissue. The monoclonalantibodies of the invention, which are monospecific for ferritin orbi-specific as regards ferritin and a receptor, allow these substancesto be targeted to the selected cells.

c) X is a Galenic Vector or a Prodrug.

To improve the stability and avoid biodegradation or toxic activitytowards healthy cells, the monoclonal antibody of the invention can becoupled to a liposome type vector or to cationic type emulsions alreadyused as a drug administration system (Texeira et al., (1999),Pharmaceutical Research, 16, 30-46). It can also be a cationic lipidsuch as those described by D. Deshpande et al., (1998), PharmaceuticalResearch, 15: 1340-1347. Integrating cytostatic and/or cytotoxicmolecules of the antisense RNA and DNA type into this type of vector isparticularly appropriate. However, for the reasons given above and withthe aim of increasing the targeting specificity and reducing thecytotoxicity of the different toxins used, the molecules cited in pointb) above can advantageously be carried by these vectors.

d) X is a Second Antibody,

-   -   either directed against ferritin allowing cross-linking of        several ferritin molecules;    -   or directed against an antigen for dendritic lymphoid cells,        macrophage cells or natural killer cells (such as CD20, CD30,        CD33, HNK1, CD56), to recruit said cell and activate it in the        tumour cell;    -   or directed against a receptor located on the target tumour cell        to allow cross-linking of the target cell with the ferritin.

In points a), b), c) and d) above, X can also be coupled to a carbonchain sensitive to esterases. This chain, with a length of 5 to 15carbon atoms, linear or branched, is sensitive to esterases. Couplingthe X molecule to this type of chain in therapeutic use is of particularadvantage since healthy cells have enzymatic systems, in particularesterases, in contrast to cancer cells. Thus in this implementation,only cancer cells will be labelled with antibodies coupled to thelabelled isotope; the action of the esterases in the healthy cells willlead to a leaching of the isotope into the general circulation, and thusonly cancer cells will benefit from specific and differential labelling.

The present invention also concerns a process for determining orlocalising the possible presence in an individual of a tumour or ofcancerous cells in which the product of a gene from the myc family isoverexpressed, comprising an in vitro, ex vivo or in vivo labelling ofsaid cells or said tumour with an anti-ferritin monoclonal antibody or afragment thereof coupled directly or indirectly to a substance emittinga signal, said antibody or said fragment recognising an epitope commonto acidic and basic human ferritins. In this process, the antibody ispreferably coupled to a radioactive isotope; by way of example, indium111 is particularly suitable when using the monoclonal antibodies of theinvention in immunoscintigraphy. The quality of the monoclonalantibodies of the invention as described above, namely its specificityand affinity, is important in the determination or localisation processof the invention.

The skilled person will know how to couple the mono- or bi-specificantibody to the suitable signal and how to detect the signal and/orlocalise a tumour or tumour cells. Examples that can be cited arefluorescence emitters or luminescence emitters coupled to the antibodyby any suitable method. One example of a method that is widely used isavidin/biotin coupling.

The invention also provides the product of coupling between a monoclonalantibody as described above and with characteristics of specificitytowards human H and L ferritins and an affinity of more than 10⁻⁹mole/litre, and a substance X selected from the group formed by:

-   -   beta-, gamma- or alpha-emitting radioisotopes;    -   cytotoxic A chain ricin or abrin molecules or A chain diphtheria        toxin;    -   cytolytic substances of the methotrexate, mitomycin, taxol or        adriamycin type;    -   antisense RNA and DNA.

It also concerns coupling products in which X is stabilised by aliposome or cationic emulsion type vector. It also concerns a couplingproduct in which a spacer constituted by a carboxylic 5 and 15 carbonatom chain is integrated into the coupling product and can liberate theX molecule when the coupling product is brought into the presence ofendogenous esterases.

Within the different aspects of the invention described above, it isparticularly surprising to observe that the use of isoferritinsmonoclonal antibodies or fragments thereof, if appropriate coupled to aradioactive isotope or to a cytotoxic or cytostatic substance has anapplication in diagnosis and the treatment of all of the tumours andcancer cells which overexpresses the myc gene products. Without in anyway limiting the field of application, different types of cancers forwhich this type of treatment would appear to be particularly suitableare:

a. Hodgkin's Disease:

It is already known that patients that are refractory to conventionaltreatments for the disease have been able to be treated with anantiferritin polyclonal antibody labelled with yttrium 90 (H. M.Vriesendorp et al., (1997), Cancer Supplement, December 15, vol. 80,n^(o) 12, pages 27-21).

b. Cancer of the Pancreas:

There are 8000 new cases per year of cancer of the pancreas in France.This cancer has a catastrophic prognosis in that less than 3% survive tofive years. This cancer is very chemo-insensitive; it is radio-sensitivebut radio incurable (critical organ). Current treatments are surgery, ifpossible, radiotherapy and per-operative radiotherapy. The advantage ofusing antiferritin monoclonal antibodies coupled to a radioactiveisotope, in particular yttrium 90, is that the deliverable dose ofradiotherapy is potentially multiplied by 20, accompanied by a secondarytoxicity that is very low. The consequence of targeting preventsdestruction of the radioactive isotope in the general circulation; theisotope is targeted directly at the tumours.

c. Hepatocellular Carcinoma:

This liver cancer represents about 15 cases per 100000 inhabitants. Itis operable in 20% of cases with a prognosis of 40% at five years. Forthe 80% of inoperable cases, the prognosis is 10% at 5 years Notreatment has yet proved effective apart from radioactive lipiodol.Treatment using the compounds of the invention can increase thetherapeutic index (residence time/discharge time) from 4 to 10 withrespect to treatment with iodine-131 labelled lipiodol, also because theisotope is administered directly to the tumour.

d. Carcinoma of the Head and Neck (Upper Aerodigestive Tract Cancers)

These cancers affect 12000 patients per year in France and have a 40%survival rate at 5 years; 15% of the survivors have a high risk ofmetastatis.

All of the cancers cited above produce ferritin. Further, while ferritinis expressed only intracellularly in healthy cells, in the neoplasticprocess, usually associated with the c-myc oncogene, ferritin isgenerally expressed on the cell membrane. It thus appears clear thatproviding a coupling product between monoclonal antibodies and anantiferritin molecule X such as that defined above can specificallytarget a wide variety of tumours or tumour cells, which all hyperexpressa gene from the myc family. In the case of a treatment with metabolicradiotherapy, the use of an IgM is advantageous because of its slowdiffusion and longer lifetime.

The above list of tumoral diseases is not exhaustive. Neuroblastomas,lung or ovarian cancers also exhibit an overexpression of c-myc.

The accompanying figures illustrate the invention and its performancewithout in any way limiting it.

LEGEND TO FIGURES

FIG. 1 shows photographs of three histochemical sections treated asindicated below and labelled with AMB 8LK antiferritin monoclonalantibodies.

Photographs A and B are sections of ganglions from Hodgkin's disease.Photograph C is a section of pancreatic adenocarcinoma.

FIG. 2 represents the chromatogram obtained after purification onSuperdex 200-26/60 column using the purification protocol described inTable II below.

FIG. 3 shows the results of a sandwich ELISA test (AMB8 LK on solidphase, AMB 8LK labelled with alkaline phosphatase in human serum).

EXAMPLE 1 Producing Antiferritin Monoclonal Antibodies

a) As stated above, it is important that the antigen used to immunisethe cells is obtained in a high degree of purity. The original techniqueused is described in table II below. TABLE II Standardisation andimmunisation of mice Human Minkowsky- Slice and grind Chauffard spleenSonicate 20 min (setting C, constant/G40) Sonicated spleen Heat to70-75° C. for 10 min Ferritin-enriched spleen Centrifuge 25000 g for 10min extract Centrifuge supernatant +acetic acid - pH 4.8 contact 2 hoursat 4° C. +K2HPO4 = pH 6.5 Centrifuge supernatant Fractionalprecipitation 50% ammonium sulphate overnight, 4° C. centrifuge 2500 gfor 10 min Centrifuge residue Take up in 100 mM K2HPO4 buffer, pH 7.2Dialysis in 100 mM K2HPO4 buffer, pH 7.2 Overnight, 4° C. Dialysissupernatant Re-precipitation from 5% cadmium sulphate Overnight at 4° C.Centrifuge at 10000 g for 30 min Centrifuge residue Take up in 100 mMK2HPO4 buffer, pH 7.2 Dialysis in 100 mM K2HPO4 buffer, pH 7.2Overnight, 4° C. Dialysis supernatant Chromatography on SUPERDEX 200Concentration by AMICON ultrafiltering 100 mM phosphate buffer, pH 7.2Ferritin 50 with Physico-chemical and immunochemical cadmium sulphateanalyses (QC)

An analysis of the chromatogram after chromatography on SUPERDEX 200 isshown in FIG. 2.

b) Mouse Immunisation:

The production and cloning of monoclonal antibodies by mouseimmunisation was carried out using the conventional Köhler and Milsteinmethod.

c) Production and Purification of Monoclonal Antibodies Produced byHybridomas:

The hybridomas were grown in an in vitro cell culture system onmultitrays (Nunc, reference 16795).

The cells were cultured in RPMI 4+TCH. The innoculum was constituted by5×10⁷ in 200 ml of TCH. RPMI medium (about 100 to 200 ml) was addedevery other day until confluence. The supernatant was harvested afterabout a week when the cells had died.

The supernatant was concentrated ten times then loaded onto achromatography column carrying protein G sepharose FF (Pharmacia), afterdiluting by half in 20 mM sodium phosphate buffer, pH 7. Afterchromatographing, the antibody was eluted with a 0.1 M HCl glycinebuffer, H 7, and the column was immediately desalted against 0.2 Mpotassium phosphate buffer, pH 7.2. The purity obtained was at least95%. The isotype, the isoelectric point and the reactivity byimmunodiffusion were then studied using these purified preparations.

Of the different antibodies obtained and shown in Table I above,monoclonal antibodies B8 and M29 appear to be of the greatest interestin terms of affinity and specificity for human cells. These twoantibodies are of isotype IgG 1 Kapa.

Histochemical Results:

Histochemistry experiments were carried out with a B8 monoclonalantibody coupled to peroxidase.

a) on Hodgkins Ganglion;

b) on pancreatic adeno-carcinoma. An immunohistochemical study wascarried out with AMB 8LK antibody diluted to 1/250^(th) in PBS bufferusing the conventional alkaline phosphatase technique.

The tissues were fixed in a bath of 4% paraformaldehyde, 0.1 Methenolamide pH 7.4 and cast into paraffin. 5μ sections were cut. Afterdewaxing in xylenethanol baths, the non specific reactions were blockedby incubating the preparation with foetal calf serum. The section wasincubated with AMB 8LK antibodies in a 1/25^(th) dilution for one hourat ambient temperature. The fixed AMB 8LK was revealed with a peroxidasesystem (Cordell J-L et al., J. Histochem 1984 32: 219-229).

The results obtained are shown in FIG. 1.

Observation of photographs A and B indicate that all of the ReedStomberg tumour cells are positive for ferritin expression.

Observation of photograph C also shows that the tumour itself is highlypositive for ferritin expression.

EXAMPLE 2 Results of Sandwich ELISA Test

Recognition of repetitive epitopes that are common to acidic and basichuman isoferritins can improve the diagnostic and therapeutic propertiesof antibodies.

The sandwich ELISA test can identify antibodies directed againstrepetitive epitopes. The principle of the test resides in fixing thesame antigen firstly with a first antibody fixed on the solid phase andsecondly with a second free antibody carrying a label (for exampleenzymatic). For the test to function, at least two antibodies mustrecognise the same molecule; in other words, the recognised moleculecomprises at least two repeated epitopes.

The monoclonal antibody of the invention AMB 8LK was used to carry out asandwich ELISA test to detect human ferritins.

More precisely, the ELISA test was carried out using the followingprotocol:

a) Incubation:

50 μl of a standard or of patient's serum was deposited in each well.200 μl of AMB 8LK antiferritin antibody conjugated with peroxidase wasadded. Beads coated with AMB 8LK antibody were then added to each well.The mixture was incubated for two hours with stirring at ambienttemperature. It was then washed three times with a solution of 1.5 ml of9% NaCl and 2% Tween 20.

b) Colour Reaction:

300 μl of substrate (OPD) was deposited in each tube. The mixture wasincubated for 30 minutes at ambient temperature in the absence of light.2 ml of 1M HCl was added to each tube.

c) Reading:

The absorbance was measured at 492 nm, reflecting the quantity ofantibody fixed to the ferritin, itself fixed to the antibody on thesolid phase. FIG. 3 shows the results of the test. It could detect humanferritin of the following origins:

-   -   spleen;    -   liver;    -   heart    -   placenta.

The results shown here show that it is possible to carry out a sandwichtest with AMB 8LK antibody. These results demonstrate that the antibodyrecognises a common repetitive epitope on human isoferritins.

1. An antiferritin monoclonal antibody or fragment thereof for themanufacturing of a diagnostic test or of a therapeutic drug for canceror tumour cells in which overexpression of a gene from the myc family isobserved with at least the following characteristics: it recognises anepitope that is repetitive to and common acidic and basic humanferritins; its affinity for the antigen is more than 10⁻⁹ mole/litre. 2.An antibody according to claim 1, of isotype IgG K or a derivativethereof.
 3. An antibody according to claim 1, of isotype IgM.
 4. Acoupling product between an antibody and a substance X selected from thegroup formed by: Beta-, alpha- or gamma-emitting radioisotopes; toxic Achain ricin or abrin type molecules, or A chain diphtheria toxin;cytolytic substances of the methotrexate, mitomycin, taxol or adriamycintype; antisense RNA; said antibody being an antiferritin monoclonalantibody recognising an epitope that is common to acidic and basic humanferritins and having an affinity for the antigen of more than 10⁻⁹mole/litre.
 5. A coupling product according to claim 4, in which saidepitope that is common to acidic and basic human ferritins isrepetitive.
 6. A coupling product according to claim 4, in which X isstabilised by a liposome type vector.
 7. A coupling product according toclaim 4, in which a C5 to C15 spacer is integrated into the couplingproduct and can liberate the X molecule when the coupling product isbrought into the presence of esterases.